Genetic dissection of the zebrafish retinal stem-cell compartment

In a large-scale forward-genetic screen, we discovered that a limited number of genes are required for the regulation of retinal stem cells after embryogenesis in zebrafish. In 18 mutants out of almost 2000 F2 families screened, the eye undergoes normal embryonic development, but fails to continue growth from the ciliary marginal zone (CMZ), the post-embryonic stem-cell niche. Class I-A mutants (5 loci) display lower amounts of proliferation in the CMZ, while nearly all cells in the retina appear differentiated. Class I-B mutants (2 loci) have a reduced CMZ with a concomitant expansion in the retinal pigmented epithelium (RPE), suggesting a common post-embryonic stem cell is the source for these neighboring cell types. Class II encompasses three distinct types of mutants (11 loci) with expanded CMZ, in which the progenitor population is arrested in the cell cycle. We also show that in at least one combination, the reduced CMZ phenotype is genetically epistatic to the expanded CMZ phenotype, suggesting that Class I genes are more likely to affect the stem cells and Class II the progenitor cells. Finally, a comparative mapping analysis demonstrates that the new genes isolated do not correspond to genes previously implicated in stem-cell regulation. Our study suggests that embryonic and post-embryonic stem cells utilize separable genetic programs in the zebrafish retina.     

Small scale application and assessment of a Index of Biotic Integrity for a large boreal river

Modifications and use of Karr’s Index of Biotic Integrity (IBI) for assessing the effects of anthropogenic impacts to aquatic ecosystems have typically occurred using data collected at the macro scale. However, some non-point sources of habitat degradation occur at small scales. One possible source of perturbation to fish habitat in boreal rivers is the application of rip rap shoreline armouring in human use areas. In this study we assess the use of IBI in a small scale setting and discuss the potential impact of rip rap shoreline armouring. We captured small and juvenile fishes weekly during 2002–2003 from 12 sample sites within the littoral zone of a human use area using a modified beach seine. Principal component analysis (PCA) was performed on the data to examine the relationship between species composition and IBI scores. We also performed PCA on the IBI metrics to assess our modifications. The IBI method produced higher scores for armoured sites than for unarmoured sites. We found a strong rank order correlation (Spearman’s ρ > 0.93; p < 0.001) between the modified IBI scores and the first principal component, suggesting that Karr’s original empirically-based IBI is strongly linked to species composition. We found a high degree of redundancy between the metrics of the IBI that validate our modifications. These results suggest that IBI can be a suitable method for assessing non-point impacts from within a small study area. Our results also indicate that fish habitat from rip rap armoured sites within the test area had consistently higher IBI scores that unarmoured sites.     

Behavioural Reproductive Isolation in a Rotifer Hybrid Zone

A hybrid zone between two Brachionus plicatilis rotifer mitochondrial DNA (mtDNA) lineages was recently described in the Iberian Peninsula between a pond (Santed 2) and a lake (Gallocanta). The patterns of mitochondrial and nuclear genetic variation observed suggested that gene flow is mainly male-mediated from the lake to the pond. Here we test two hypotheses: (a) that male-mediated gene flow occurs through assortative mating between individuals from these ponds, (b) that behavioural isolation occurs between the two mtDNA lineages. We isolated, reared and genotyped rotifer clones from resting eggs collected in the sediments of these and two other distant ponds. We devised a quick, inexpensive RFLP method to discriminate between B. plicatilis and its sibling species B. ‘Manjavaeas’ and between both mtDNA B. plicatilis lineages. Behavioural no-choice tests using new-born, virgin males and females were performed between five clones. B. ‘Manjavacas’ and B. plicatilis were reproductively isolated. B. plicatilis clones did not show evidence of reproductive isolation, regardless of their mtDNA lineage, except Santed 2 males, which discriminated strongly against Gallocanta females. These results could help to explain the discrepancies between mitochondrial and nuclear genetic variation reported in the two populations.     

Temporally regulated expression of Cre recombinase in neural stem cells

The use of mouse gene targeting to study molecules important in neural development is oftentimes impaired by early embryonic lethality. In order to address later roles for such molecules, specifically in neural stem cells, we generated transgenic mice that express both the tetracycline-inducible molecule rtTA-M2 and GFP under the control of the neural precursor specific form of nestin. Developmental analysis of these mice demonstrates that GFP expression is exclusive to the neural tube. Adult expression of GFP is seen only in known areas of adult neurogenesis, namely, the subventricular zone and the dentate gyrus. When crossed with a second transgenic mouse (TetOp-Cre) that expresses the Cre recombinase under the control of the tetracycline responsive promotor, we demonstrate temporal induction of Cre in bigenic animals exposed to doxycycline. We further demonstrate the feasibility of this approach by using the ROSA-26 reporter mouse to mediate recombination in neural precursor cells.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.     

Determination of protein charge by capillary zone electrophoresis

The feasibility of employing classical electrophoresis theory to determine the net charge (valence) of proteins by capillary zone electrophoresis is illustrated in this paper. An outline of a procedure to facilitate the interpretation of mobility measurements is demonstrated by its application to a published mobility measurement for Staphylococcal nuclease at pH 8.9 that had been obtained by capillary zone electrophoresis. The significantly higher valence of +7.5 (cf. 5.6 from the same series of measurements) that has been reported on the basis of a "charge ladder" approach for charge determination signifies the likelihood that the latter generic approach may be prone to error arising from nonconformity of the experimental system with an inherent assumption that chemical modification or mutation of amino acid residues has no effect on the overall three-dimensional size and shape of the protein.     

Reconstructing asymmetrical reproductive character displacement in a periodical cicada contact zone

Selection against costly reproductive interactions can lead to reproductive character displacement (RCD). We use information from patterns of displacement and inferences about predisplacement character states to investigate causes of RCD in periodical cicadas. The 13-year periodical cicada Magicicada neotredecim exhibits RCD and strong reproductive isolation in sympatry with a closely related 13-year species, Magicicada tredecim. Displacement is asymmetrical, because no corresponding pattern of character displacement exists within M. tredecim. Results from playback and hybridization experiments strongly suggest that sexual interactions between members of these species were possible at initial contact. Given these patterns, we evaluate potential sources of selection for displacement. One possible source is 'acoustical interference', or mate-location inefficiencies caused by the presence of heterospecifics. Acoustical interference combined with the species-specificity of song pitch and preference appears to predict the observed asymmetrical pattern of RCD in Magicicada. However, acoustical interference does not appear to be a complete explanation for displacement in Magicicada, because our experiments suggest a significant potential for direct sexual interactions between these species before displacement. Another possible source of selection for displacement is hybrid failure. We evaluate the attractiveness of inferred hybrid mating signals, and we examine the viability of hybrid eggs. Neither of these shows strong evidence of hybrid inferiority. We conclude by presenting a model of hybrid failure related to life cycle differences in Magicicada.     

Sequence Representation and Prediction of Protein Secondary Structure for Structural Motifs in Twilight Zone Proteins

Characterizing and classifying regularities in protein structure is an important element in uncovering the mechanisms that regulate protein structure, function and evolution. Recent research concentrates on analysis of structural motifs that can be used to describe larger, fold-sized structures based on homologous primary sequences. At the same time, accuracy of secondary protein structure prediction based on multiple sequence alignment drops significantly when low homology (twilight zone) sequences are considered. To this end, this paper addresses a problem of providing an alternative sequences representation that would improve ability to distinguish secondary structures for the twilight zone sequences without using alignment. We consider a novel classification problem, in which, structural motifs, referred to as structural fragments (SFs) are defined as uniform strand, helix and coil fragments. Classification of SFs allows to design novel sequence representations, and to investigate which other factors and prediction algorithms may result in the improved discrimination. Comprehensive experimental results show that statistically significant improvement in classification accuracy can be achieved by: (1) improving sequence representations, and (2) removing possible noise on the terminal residues in the SFs. Combining these two approaches reduces the error rate on average by 15% when compared to classification using standard representation and noisy information on the terminal residues, bringing the classification accuracy to over 70%. Finally, we show that certain prediction algorithms, such as neural networks and boosted decision trees, are superior to other algorithms.This research was supported in part by the Natural Sciences and Engineering Research Council of Canada (NSERC).     

A gradient analysis based on the buffer zones of urban landscape pattern of the constructed area in Guigang City, Guangxi, China

Over the last century urbanization on the landscape has increased and intensified. Urban development has a great impact on the environment at the local, regional and even global levels. As a driving force in global change, the need to understand the dynamics of urban pattern and its change in an accurate and efficient manner is ever more pressing. Based on aerial color infrared photography in 1985 and QuickBird satellite imagery in 2004 and according to the standard for plan and construction of city land use, the landscape of Guigang City was divided into 11 types. In the landscape classification maps, 31 buffer zones, each being 200 m wide, were divided. With the aid of GIS software ArcView 3.2 and landscape pattern analysis software FRAGSTATS 3.3, the landscape spatial patterns of each buffer zone were analyzed at the landscape level and class level. The landscape indices, such as patch size, patch fractal dimension, diversity index and evenness index, were calculated. The results indicated the following: The total area of the residential land and the communal land in 2004 covered 46.3% of the entire constructed area of Guigang City and the sum of the patch number of the two patch types occupied about 39.7% of the total patch number, while the percentages were respectively 48.2% and 45.4% in 1985. This showed that the mosaic landscape with the residential land and communal facilities land became increasingly unclear following urban development and landscape diversity. Based on the gradient analysis with the landscape-level index and the class-level index, there were two business and finance centers in the constructed area. One was located in the urban center with a range of 0.8 km; the other was from the 10^th to the 16^th zone. In each buffer zone, the mean patch size was larger and the landscape shape was more regular in 2004 than in 1985. Furthermore, the Shannon diversity index of each buffer zone rose in 2004 with patch richness and evenness increasing. The landscape index computed for the main landscape types (communal facilities land, industrial estate land, residential land and farmland) in the middle of the buffer zones clearly indicated the changes taking place in urbanization. The patch size and the patch number of industrial estate land and farmland also pointed to these changes, while communal facilities land and residential land assumed another similar tendency towards changes. In each gradient zone, industrial estate land had the most complex shape, the lowest area percentage of each zone and the biggest mean patch size, whereas communal facilities land and residential land were opposite to industrial estate land. Farmland had a steady percentage along the buffer zone and its continuous distribution had an important impact on the urban eco-environment. The question of how to relate the pattern of changing characteristics along the buffer zone to the urban ecological process and urban ecological planning remains to be studied further in the future.